If you were to start up a molecular biology lab today, would you require a setup for radioactive work? With all the various chromogenic, chemiluminescent and fluorogenic detection reagents available today, is radioactivity extraneous? Any specific protocols still absolutely requiring radioactivity? Thanks, Curious John
Hi John, Speaking as a practising molecular biologist, I can think of no routinely used procedure performed today involving radioactive molecules. In fact, most if not all of the older hazardous reagents that were used a decade ago are easily replaceable now. Many mutagenic compounds like EtBr can be replaced with harmless and less cumbersome to manage alternatives, though equipment used to visualise the results may need to be updated to maintain resolution. One use of Radioactivity that used to be commonplace was in-situ hybridisations and protein or DNA blots, but these have been entirely replaced with flourescent alternatives by now. The only valid use of Radioactivity I've encountered that remains in reasonably mainstream use is mutation breeding for plants or the like, but even in this case one can use an X-ray machine instead of a radioactive source. I hope this helped! If you have questions about replacing particular reagents or protocols with less radioactive alternatives, I'd be happy to help look it up for you. -Cathal Garvey
Although I don't use radioactivity often, there are still a few procedures where I do or would choose to use it. For blotting/hybridization procedures, I would still choose radioactivity, hands down. As far as I know, most non-radioactive detection procedures require an immunoblot-like detection step. In my opinion, this just adds one more additional set of steps that needs to be optimized and opportunity for noise or background to be introduced inot the experiment. When might one want to do some type of hybridization experiment? A conventional library screen (I've done one of these in the last 2 years), outhern bolt to determination of the number of loci for a transgenic event, or looking at whether there are multiple classes of transcripts corresponding to a given gene. For the latter, if one wants quantitative data, radioactivity is linear over several orders of magnitude while most non-radioactive detection systems have a far more limited linear range. Of course there are many biochemistry experiments that are most easily done with radioactivity, but this will be largely dependent on exactly what you are working on. I may be facing doing kinetics on a methyl transferase soon. From my perusal of the literature, this looks like it will be much simpler to do with radioactivity than by any other approach. Mike On Dec 10, 2009, at 5:43 PM, Hachey, John wrote: > > If you were to start up a molecular biology lab today, would you(Continue reading)
In article <mailman.35.1260489668.13172.methods@...>, "Hachey, John" <John.Hachey@...> wrote: > >If you were to start up a molecular biology lab today, would you require >a setup for radioactive work? With all the various chromogenic, > chemiluminescent and fluorogenic detection reagents available today, >is radioactivity extraneous? Any specific protocols >still absolutely requiring radioactivity? "Absolutely" - probably no or very little. "In reality" - there are plenty of things that are done much more directly, more reliably and less expensively with isotopes. Driving car to work is many orders of magnitude more dangerous to your health than using isotopes in the lab. Forsaking direct work for the poorly reproducible mega expensive garbage kits because of the irrational fear of radioactivity makes absolutely no sense. Every time I see those "quantitations" of protein phosphorylation by mass-spec or by indirect immunoassays, I can't stop laughing. DK
Hi, I would agree... GST pull downs just didn't work using the antibodies I had... a bit of 35S and it all went fine. In our lab there is very little radioactive work and if you look at the amounts used the risk is very low. Take the 35S methionine i used for example: The ALI (annual limit on intake) for this isotope is 10 mCi. I used in total for all my experiments less than 1 mCi. As I was not eating the stuff, worked with gloves and tried to be reasonably careful, I assume not to have incorporated any radiation, but even if I had 'eaten' the entire aliquot I would still only have doubled my annual background radiation, as it would not even amount to 10% ALI. 32P is of course a stronger source and actually requires adequate shielding, but still you have a dosimeter monitoring whether or not you have been overexposed. In summary: I would set up a lab with radiation facilities, however nevertheless try and reduce radioactive work where sensibly possible. All The Best! > -- -- GRATIS für alle GMX-Mitglieder: Die maxdome Movie-FLAT! Jetzt freischalten unter http://portal.gmx.net/de/go/maxdome01(Continue reading)
A added a new section on Protein display/Protein engineering/Directed evolution protocols. Details: http://www.sciclips.com/sciclips/bio-protocols.do
Hi Folks, Anyone any idea why if one autoclaves 10% (w/v) SDS prepared in MilliQ water for 25mins instead of 15mins, it will no longer go back into solution even when diluted 100fold into warm water? Biorad is the source a. If you know how to get it back into solution, please tell. I've never come across this before. Most odd. Duncan -- -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd.
Hello, I am trying to find the kd for my protein which binds GTP. I have done two sets of experiments: 1) taken constant protein and increased unlabeled GTP and monitored the tryptophan fluorescence quenching. 2) constant labeled (mant)-GTP and increased the protein and monitored the fluorescence increase with increasing protein. I want to know if someone can help me and tell the equations (and the formulas) to feed these values in , in order to calculate the kd. Thanks Ruchi
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